Binding Protein Concentrations Research Articles

Small volume rapid equilibrium dialysis (RED) measures effects of interstitial parameters on the protein-bound fraction of topical drugs

The importance of plasma protein binding in the early stages of drug development is well recognized. Free and bound drug fractions in plasma are routinely determined with well-established methods. However, for physiological fluids with a small accessible volume and low protein concentrations, such as dermal interstitial fluid (dISF) validated methods are currently missing. Due to the low protein concentration and highly dynamic processes in the dermis, protein binding data obtained from plasma samples may underestimate in-vivo efficacy. This study aimed to validate a small volume rapid equilibrium dialysis (RED) for low protein samples, as a tool to examine drug-protein binding directly in the biological fluid at the site of action. The sample volume required for RED was successfully downscaled to 50 µl and plasma protein binding values of the four model drugs were consistent with previous studies with an average recovery of 88 ± 8% which makes all tested drugs suitable for small volume RED. Inter- and intra-batch variability showed sufficient reproducibility across RED plates. Small volume RED was successfully applied to assess the effects of interstitial parameters, including the evaluation of the major binding protein and the effects of binding protein concentration, drug concentration, and pH on the protein-bound drug fraction using 2% HSA and/or diluted human plasma as a surrogate for dISF.

Changes in Skeletal Muscle Troponin T and Vitamin D Binding Protein (DBP) Concentrations in the Blood of Male Amateur Athletes Participating in a Marathon and 100 km Adventure Race

This study assessed changes in creatine kinase (CK) activity and skeletal muscle troponin T (sTnT) concentrations in the blood, to estimate the degree of muscle degradation after exercise. In addition, the concentration of vitamin D binding protein (DBP) in the blood was assessed. DBP concentrations were measured in blood as a marker for plasma load by monomeric actin. The study included marathon (MR) participants and 100 km adventure race (AR) participants, who were examined before and after the race. There was a significant (16-fold) increase in CK activity among AR participants, and a significant increase in sTnT concentration-127% in the MR group and 113% in the AR group, while there was a statistically significant decrease in DBP concentration by 14% in the AR group. In addition, it was observed that the initial concentration of DBP in both groups was in a normal range, but was lower than the average population, and the DBP concentration in the AR group was lower than in the MR group. It was concluded that exhausting physical effort such as a marathon or adventure races causes muscle damage with a far stronger influence on sarcoplasm than on filaments. The short-term and slight reduction in the concentration of DBP in blood after such efforts may be due to the appearance of monomeric actin in plasma.

MicroRNA-483-3p Inhibitor Ameliorates Sepsis-Induced Intestinal Injury by Attenuating Cell Apoptosis and Cytotoxicity Via Regulating HIPK2.

Sepsis is a life-threatening syndrome that can result in multi-organ dysfunction. MicroRNA (miR)-483-3p was previously demonstrated to be upregulated in sepsis patients; however, its specific functions in sepsis-triggered intestinal injury remain unclarified. Human intestinal epithelial NCM460 cell line was stimulated with lipopolysaccharide (LPS) to mimic sepsis-induced intestinal injury in vitro. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was utilized for examining cell apoptosis. Western blotting and real time quantitative polymerase chain reaction (RT-qPCR) were used for detecting molecular protein and RNA levels. LPS-induced cytotoxicity was determined by measuring concentrations of lactate dehydrogenase (LDH), diamine oxidase (DAO) and fatty acid binding protein 2 (FABP2). Luciferase reporter assay was utilized for verifying the interaction between miR-483-3p and homeodomain interacting protein kinase 2 (HIPK2). Inhibiting miR-483-3p alleviates LPS-triggered NCM460 cell apoptosis and cytotoxicity. miR-483-3p targeted HIPK2 in LPS-stimulated NCM460 cells. Knockdown of HIPK2 reversed the above effects mediated by miR-483-3p inhibitor. Inhibiting miR-483-3p ameliorates LPS-triggered apoptosis and cytotoxicity by targeting HIPK2.

You are What You Drink? How Associations Between Profiles of Beverage Consumption and Type 2 Diabetes Risk are Mediated by Biomarker Networks

BackgroundMultiple studies have independently investigated the associations of the consumption of individual beverage types and specific plasma biomarkers with the risk of type 2 diabetes (T2D). However, as individuals do not consume single beverage types exclusively and plasma biomarkers do not act in isolation, it remains unclear how patterns of beverage consumption and plasma biomarker networks associate both with each other and T2D risk. ObjectivesWe aimed to elucidate potential dietary determinants of T2D risk by defining a model that describes habitual beverage consumption profiles in relation to identified networks of circulating plasma biomarkers. MethodsThis study included 1,461 case and 1,568 control participants from case-control studies of T2D nested within the Nurses’ Health Study. Participants completed validated semiquantitative food frequency questionnaires that assessed habitual beverage consumption, and they provided blood samples from which 27 plasma biomarkers of cardiometabolic risk were identified. Common exploratory factor analysis (EFA) identified factors that separately described beverage consumption profiles and biomarker networks. Multivariable-adjusted regression elucidated the relationships between beverage and biomarker factors and T2D risk. ResultsEFA revealed five factors describing unique beverage consumption profiles and seven factors describing biomarker networks. The factor describing alcoholic beverage consumption was associated with a reduced risk of T2D (odds ratio [OR]: 0.50 [0.40, 0.64], P<0.001) mediated, in part, by the factor describing increased concentrations of adiponectin biomarkers (19.9% [12.0, 31.1] P = 0.004). The factor describing low-calorie sweetened beverage (LCSBs) consumption was associated with an increased risk of T2D (OR: 1.33 [1.03, 1.72], P = 0.021), and the factor describing lower concentrations of insulin-like growth factor binding proteins 1 and 2, and soluble leptin receptor, and increased leptin concentrations (P = 0.005). ConclusionsModerate alcohol consumption was associated with reduced T2D risk, mediated in part by increased circulating adiponectin. LCSB consumption was associated with both increased T2D risk and perturbed insulin-like growth factor and leptin signaling.

Abstract 5544: Association of circulating markers of gut barrier damage and inflammation with colorectal neoplasia stage

Abstract A disturbance in the microbiome facilitated by gut barrier dysfunction and inflammation can influence the progression of colorectal neoplasia. One of the most studied indicators of gut barrier status is lipopolysaccharide (LPS). Increased permeability of the gut barrier leads to extensive translocation of microbes into the lamina propria, which is associated with higher circulating levels of LPS. Additionally, increased serum concentrations of inflammatory cytokines including IFNy, IL6, IL10, and TNFα have been associated with impaired gut integrity and its progressive dysfunction. We investigated the role of gut barrier permeability and inflammation in colorectal cancer (CRC) development by assessing markers of intestinal barrier dysfunction and inflammation in two different patient cohorts; Irish (discovery) and Czech (validation). ELISA assays were used to assess circulating concentrations of lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP) in serum from patients with CRC (n=30), colorectal adenoma (n=40), and negative after colonoscopy/blood donor controls (n=35). Circulating levels of inflammatory cytokines and calprotectin were assessed using an MSD MultiSPOT platform and an MSD R-plex assay (Mesocale Discovery, Rockville, Md, United States), respectively. Differences between pathology groups were assessed using the Kruskal Wallis or Mann-Whitney U test. Receiver operating curve (ROC) analysis was implemented to identify single and combinations of biomarkers capable of discriminating between CRC patients and controls with maximum sensitivity (SE) and specificity (SP) (RStudio, PBC, Boston, MA URL http://www.rstudio.com/). In the discovery cohort, levels of LBP, TNFα, IL6, calprotectin and CRP were significantly higher in CRC patients than in controls after false discovery rate correction (p=0.02, p=0.03, p=0.006, p=0.01, p=0.006, respectively). The area under the ROC curve (AUC) was 0.76 (95% CI: 0.64-0.87, p = 0.003) for LBP, 0.69 (95% CI: 0.55-0.82, p = 0.007) for calprotectin and 0.87(95% CI: 0.74-1.00, p = 0.003) for CRP. The optimal biomarker combination for discriminating between CRC and controls was the combination of the three markers (SP= 0.75, SE = 0.75). In the validation cohort, levels of calprotectin and TNFα, were significantly higher in cancer compared to control (p=0.05, p=0.02, respectively). Furthermore, IFNγ and IL10 were also significantly elevated in cancer compared to control (p= 0.02, p=0.003, respectively). These results support the hypothesis that a dysfunctional gut barrier and attendant inflammation may influence CRC development and progression. Additionally, LBP, CRP and calprotectin may have potential as diagnostic biomarkers for CRC or for risk stratification. Gut bacterial and barrier wall modulation and stabilization has potential for preventing colorectal adenomas and hence colorectal cancer. Citation Format: Flavia Genua, Petr Holý, Pavel Souček, Václav Liška, David J. Hughes. Association of circulating markers of gut barrier damage and inflammation with colorectal neoplasia stage. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5544.

Maternal vitamin D status and attention deficit hyperactivity disorder (ADHD), an under diagnosed risk factor; A review

Vitamin D is important to mediate several brain processes such as proliferation, apoptosis, and neurotransmission in early stages of life. Vitamin D deficiency during critical periods of development can lead to persistent brain alterations. Vitamin D homeostasis during pregnancy is affected by two factors which includes an increase in mother’s calcitriol levels and an increase in mother’s Vitamin D Binding protein concentrations. Attention deficient hyperactivity disorder (ADHD) is an outcome of a complicated interaction between genetic, environmental, and developmental traits, and genetic factors cover about 80% of the cases. The efficiency of the immune system can be altered by a deficiency of Vitamin D in maternal body and maternal stress during gestation such as perinatal depression. Studies have proved that during gestation if there is a deficiency of vitamin D in maternal body, it can influence the brain development of the fetus and can also alter the synthesis of the brain-derived neurotropic factor. The current manuscript has been compiled to elaborate different factors which are associated with ADHD particularly focusing on the relationship of vitamin D deficiency in mothers. References material was selected from NCBI (PUBMED), Science direct, Google scholar, Publons etc. Using the terms ADHD, Vitamin D and Maternal nutritional status. Although, controversial relationship was found between the deficiency of Vitamin D level in pregnant women and development of ADHD in children but more controlled trials are required for future direction as well as to rule out other associated causes.

Tcf7l2 in hepatocytes regulates de novo lipogenesis in diet-induced non-alcoholic fatty liver disease in mice

Aims/hypothesisNon-alcoholic fatty liver disease (NAFLD) associated with type 2 diabetes may more easily progress towards severe forms of non-alcoholic steatohepatitis (NASH) and cirrhosis. Although the Wnt effector transcription factor 7-like 2 (TCF7L2) is closely associated with type 2 diabetes risk, the role of TCF7L2 in NAFLD development remains unclear. Here, we investigated how changes in TCF7L2 expression in the liver affects hepatic lipid metabolism based on the major risk factors of NAFLD development.MethodsTcf7l2 was selectively ablated in the liver of C57BL/6N mice by inducing the albumin (Alb) promoter to recombine Tcf7l2 alleles floxed at exon 5 (liver-specific Tcf7l2-knockout [KO] mice: Alb-Cre;Tcf7l2f/f). Alb-Cre;Tcf7l2f/f and their wild-type (Tcf7l2f/f) littermates were fed a high-fat diet (HFD) or a high-carbohydrate diet (HCD) for 22 weeks to reproduce NAFLD/NASH. Mice were refed a standard chow diet or an HCD to stimulate de novo lipogenesis (DNL) or fed an HFD to provide exogenous fatty acids. We analysed glucose and insulin sensitivity, metabolic respiration, mRNA expression profiles, hepatic triglyceride (TG), hepatic DNL, selected hepatic metabolites, selected plasma metabolites and liver histology.ResultsAlb-Cre;Tcf7l2f/f essentially exhibited increased lipogenic genes, but there were no changes in hepatic lipid content in mice fed a normal chow diet. However, following 22 weeks of diet-induced NAFLD/NASH conditions, liver steatosis was exacerbated owing to preferential metabolism of carbohydrate over fat. Indeed, hepatic Tcf7l2 deficiency enhanced liver lipid content in a manner that was dependent on the duration and amount of exposure to carbohydrates, owing to cell-autonomous increases in hepatic DNL. Mechanistically, TCF7L2 regulated the transcriptional activity of Mlxipl (also known as ChREBP) by modulating O-GlcNAcylation and protein content of carbohydrate response element binding protein (ChREBP), and targeted Srebf1 (also called SREBP1) via miRNA (miR)-33-5p in hepatocytes. Eventually, restoring TCF7L2 expression at the physiological level in the liver of Alb-Cre;Tcf7l2f/f mice alleviated liver steatosis without altering body composition under both acute and chronic HCD conditions.Conclusions/interpretationIn mice, loss of hepatic Tcf7l2 contributes to liver steatosis by inducing preferential metabolism of carbohydrates via DNL activation. Therefore, TCF7L2 could be a promising regulator of the NAFLD associated with high-carbohydrate diets and diabetes since TCF7L2 deficiency may lead to development of NAFLD by promoting utilisation of excess glucose pools through activating DNL.Data availabilityRNA-sequencing data have been deposited into the NCBI GEO under the accession number GSE162449 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE162449).Graphical abstract

Increased Serum Total and Free 25-Hydroxyvitamin D with Daily Intake of Cholecalciferol-Fortified Skim Milk: A Randomized Controlled Trial in Colombian Adolescents

BackgroundThe efficacy of cholecalciferol (vitamin D3) food fortification in low- and middle-income countries near the Equator is unknown. ObjectivesWe examined the effects of providing cholecalciferol-fortified skim milk to adolescents and their mothers on serum total 25(OH)D, free 25(OH)D, and vitamin D–binding protein (DBP) concentrations in a randomized controlled trial. MethodsWe randomly assigned 80 Colombian families each with a child aged 12–14.5 y and their mother 1 L of skim milk daily, either fortified with 2400 IU (60 μg) cholecalciferol or unfortified, for 6 wk. We prescribed 500 mL of milk daily to adolescents; mothers consumed the remainder ad libitum. We estimated intent-to-treat effects as the between-arm difference in the change in serum total and free 25(OH)D and DBP concentrations from baseline to the end of follow-up. Secondary analyses included stratification by baseline characteristics and per-protocol comparisons. ResultsAmong adolescents, fortification effects (95% CI) on serum total 25(OH)D, free 25(OH)D, and DBP concentrations were 5.4 nmol/L (2.1, 8.8 nmol/L), 0.6 pmol/L (−0.2, 1.4 pmol/L), and −416 nmol/L (−944, 112 nmol/L), respectively. Effects on total 25(OH)D were stronger in adolescents with lower DBP concentrations, darker skin, less sunlight exposure, and higher compliance than in their respective counterparts. Fortification increased free 25(OH)D concentrations in high compliers. Among mothers, the effects (95% CI) on total 25(OH)D and DBP concentrations were 4.0 nmol/L (0.6, 7.5 nmol/L) and −128 nmol/L (−637, 381 nmol/L), respectively. There were no adverse events. ConclusionsProvision of cholecalciferol-fortified skim milk increases serum total 25(OH)D concentrations in Colombian adolescents and adult women.

Plasma levels of intestinal fatty-acid binding protein (I-FABP), abdominal distension and hydrogen concentration after lactitol small intestinal bacterial overgrowth (SIBO) test.

Breath tests with glucose, lactulose or lactitol are useful for diagnosis of small intestinal bacterial overgrowth (SIBO). Nevertheless, they have suboptimal sensitivity and specificity and, indeed, are positive in a considerable number of patients with irritable bowel syndrome (IBS). The complexity in the management of patients with functional intestinal disorders and the availability of these tests are leading to frequent diagnoses of SIBO. Intestinal Fatty-Acid Binding protein (I-FABP) is a protein present in the cytosol of intestinal epithelial cells. Its plasmatic levels have been related to different enteropathies and, therefore, could be a marker of early intestinal damage with unconfirmed clinical utility. Hence, we have studied the plasmatic I-FABP level of patients who are requested a lactitol test to confirm SIBO and related it to clinical and laboratory characteristics and SIBO test results.

Chac1 silencing mitigates hemorrhagic shock-induced intestinal injury by inhibiting oxidative stress and ferroptosis

Hemorrhagic shock (HS) is a common and significant cause of mortality and morbidity, often resulting in structural damage and dysfunction of the intestines. ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (Chac1) has been reported to be involved in the regulation of oxidative stress and ferroptosis in mammals. Herein, we investigate the effects of Chac1 on HS-induced intestinal injury induced by HS both in vitro and in vivo. Sprague-Dawley rat model with HS was established, and our investigations showed upregulation of the mRNA and protein levels of Chac1 in the model’s ileum tissues. Histopathological analysis revealed that knockdown of Chac1 attenuated the intestinal injury induced by HS. Depletion of Chac1 also reduced the increase in intestinal fatty acid binding protein (I-FABP) concentration. Immunofluorescence staining indicated that silencing Chac1 significantly suppressed the downregulation of occludin and zonula occludens-1 (ZO-1). HS-induced changes in lipid peroxidation (LPO), malondialdehyde (MDA), and glutathione (GSH) levels were reversed in the absence of Chac1, suggesting that downregulation of Chac1 alleviated HS-induced oxidative stress. Additionally, HS led to a decrease in glutathione peroxidase 4 (Gpx4) and ferritin heavy chain 1 (Fth1) expression, along with an increase in ferrous ion (Fe2+) concentration. Knockdown of Chac1 significantly inhibited ferroptosis by increasing Gpx4 and Fth1 expression while reducing the Fe2+ concentration. In vitro experiments using the rat small intestine crypt epithelial cells (IEC-6) demonstrated that depletion of Chac1 suppressed oxidative stress and ferroptosis induced by hypoxia/reoxygenation (H/R). In conclusion, our study provides evidence that downregulation of Chac1 mitigates HS-induced intestinal injury by inhibiting oxidative stress and ferroptosis.

Resveratrol attenuated high intensity exercise training-induced inflammation and ferroptosis via Nrf2/FTH1/GPX4 pathway in intestine of mice.

Moderate exercise has beneficial effects for human health and is helpful for the protection against several diseases. However, high intensity exercise training caused gastrointestinal syndrome. Resveratrol, a plant extract, plays a vital role in protecting various organs. However, whether resveratrol protected mice against high intensity exercise training-induced intestinal damage remains unclear. In this study, our objective was to investigate the protective effects and mechanism of resveratrol in high intensity exercise training-treated mice. Mice were treated with swimming exercise protocol and/or resveratrol (15 mg/kg/day) for 28 consecutive days. Then, the mice were sacrificed, and a series of evaluation indicators, including inflammatory factors and intestinal permeability of the gut, were measured based on this model. The expressions of inflammatory factors (tumor necrosis factor (TNF)-α; interferon (IFN)-γ, interleukin (IL)-6 and IL-10), oxidative stress (Nrf2, glutathione (GSH), hydrogen peroxide (H2 O2), catalase (CAT) and malondialdehyde(MDA)), intestinal barrier (gut permeability, ZO-1, Occludin and Claudin-1 as well as ferroptosis (Fe2+, Fe3+, SLC7A11, glutathioneperoxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1)) were measured, respectively. High intensity exercise training induced colon damage, manifested as inflammation (increased TNF-α, IFN-γ and IL-6 concentrations, and decreased IL-10 concentration), oxidative stress (the increase of H2O2 and MDA concentration, and the reduced CAT and GSH activities), intestinal barrier injury (increased gut permeability and intestinal fatty-acid binding protein concentration,and inhibited ZO-1, Occludin and Claudin-1 expressions) and ferroptosis (the increased of Fe2+ and Fe3+ concentrations, and suppressed phosphorylated Nrf2, SLC7A11, GPX4 and FTH1), which was relieved by resveratrol treatment in mice. Resveratrol attenuated high intensity exercise training-induced inflammation and ferroptosis through activating Nrf2/ FTH1/GPX4 pathway in mouse colon, which providing new ideas for the prevention and treatment of occupational disease in athlete.

Multifaceted Effects and Mechanisms of N-Acetylcysteine on Intestinal Injury in a Porcine Epidemic Diarrhea Virus-Infected Porcine Model.

This study investigates the potential effects of N-acetylcysteine (NAC) on intestinal injury in a porcine epidemic diarrhea virus (PEDV)-infected porcine model. Thirty-two piglets are randomly assigned to one of four groups: the control, PEDV, NAC, and NAC+PEDV. Piglets in the NAC+PEDV group are orally administrated with NAC (100mg(kg·BW)-1 day-1 ) for 4 consecutive days after 2 days of PEDV infection. The results show that NAC administration decreases the diarrhea rate and improves intestinal morphology. The concentration of diamine oxidase and intestinal fatty-acid binding protein, as well as IL-1β, IL-8, and TNF-α in the plasma, is decreased by NAC. Intriguingly, NAC administration significantly increases the viral load in the jejunum and ileum and down-regulates the expression of interferon-related genes. Microarray and proteomic analyses show that the differentially expressed genes/proteins between NAC+PEDV and PEDV groups are highly enriched in substance transport. Furthermore, aquaporin 8/10 expression is significantly increased by NAC upon PEDV infection. NAC administration alleviates PEDV-induced intestinal injury by inhibiting inflammatory responses and improving substance transport, but promotes viral replication by inhibiting interferon signaling. These results suggest NAC exhibits multifaceted effects upon PEDV infection, and thus caution is required when using NAC as a dietary supplement to prevent viral infection.

Reference intervals for free testosterone in adult men measured using a standardized equilibrium dialysis procedure.

Free testosterone (FT) determination may be helpful in evaluating men suspected of testosterone deficiency especially in conditions with altered binding-protein concentrations. However, methods for measuring FT by equilibrium dialysis and reference intervals vary among laboratories. To determine reference intervals for FT in healthy, nonobese men by age groups as well as in healthy young men, 19-39 years, using a standardized equilibrium dialysis procedure METHODS: We measured FT in 145 healthy, nonobese men, 19 years or older, using a standardized equilibrium dialysis method performed for 16-h at 37°C using undiluted serum and dialysis buffer that mimicked the ionic composition of human plasma. FT in dialysate was measured using a CDC-certified liquid chromatography tandem mass spectrometry assay. In healthy nonobese men, the 2.5th, 10th, 50th, 90th, and 97.5th percentile values for FT were 66, 91, 141, 240, and 309 pg/ml, respectively; corresponding values for men, 19-39 years, were 120, 128, 190, 274, and 368 pg/ml, respectively. FT levels by age groups exhibit the expected age-related decline. FT levels were negatively associated with body mass index, age, and sex hormone-binding globulin (SHBG) levels. Percent FT was lower in middle-aged and older men than young men adjusting for SHBG level. Further studies are needed to determine how these reference intervals apply to the diagnosis of androgen deficiency in clinical populations and in men of different races and ethnicities in different geographic regions. Reference intervals for free FT levels (normative range 66-309 pg/ml [229-1072 pmol/L] in all men and 120-368 pg/ml [415-1274 pmol/L] in men, 19-39 years), measured using a standardized equilibrium dialysis method in healthy nonobese men, provide a rational basis for categorizing FT levels. These intervals require further validation in other populations, in relation to outcomes, and in randomized trials.

Protein adsorption on core-shell resins for flow-through purifications: Effect of protein molecular size, shape, and salt concentration.

This work addresses the functional properties of the core-shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose-based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

Role of gut microbiota-derived branched-chain amino acids in the pathogenesis of Parkinson’s disease: An animal study

Neuroinflammation caused by the disorder of gut microbiota and its metabolites is associated with the pathogenesis of Parkinson's disease (PD). Thus, it is necessary to identify certain molecules derived from gut microbiota to verify whether they could become intervention targets for the treatment of PD. The branched-chain amino acids (BCAAs), as a common dietary supplement, could modulate brain function. Herein, we investigated the longitudinal shifts of microbial community in mice treated with rotenone for 0, 3 and 4 weeks by 16S rRNA gene sequencing to identify the microbial markers at different PD stages. Serum BCAAs were determined by gas chromatography-mass spectrometry. Then, rotenone-induced mice were given a high BCAA diet to evaluate the motor and non-motor functions, dopaminergic neuron loss, and inflammation levels. Using a PD mouse model, we discovered that during PD progression, the alterations of gut microbiota compositions led to the peripheral decrease of BCAAs. Based on the serum lipopolysaccharide binding protein concentrations and the levels of pro-inflammatory factors (including tumor necrosis factor-α, interleukin [IL]-1β, and IL-6) in the colon and substantia nigra, we found that the high BCAA diet could attenuate the inflammatory levels in PD mice, and reverse motor and non-motor dysfunctions and dopaminergic neuron impairment. Together, our results emphasize the dynamic changes of gut microbiota and BCAA metabolism and propose a novel strategy for PD therapy: a high BCAA diet intervention could improve PD progression by regulating the levels of inflammation.

Detection of Bacterial DNA in the Plasma of Psoriasis Patients and its Association with Serum Intestinal Fatty Acid Binding Protein Concentration

Background: There is evidence of the role of microorganisms, in particular in the throat and skin in psoriasis pathogenesis. This study aimed to investigate the presence of bacterial DNA (bacDNA) in patients' plasma and its association with gut barrier integrity . Methodology: Plasma was collected from 60 psoriasis patients in the flare up stage and 40 healthy controls. BacDNA was detected by PCR amplification using a 16S ribosomal DNA primer. The amplified DNA fragments were then sequenced to determine their bacterial origin. Results: Bacterial DNA was detected in 51 of 60 patients and only in 4 of 40 healthy controls. BacDNA sequencing results revealed that these bacterial species originated from the gut and skin. On the other hand, intestinal fatty acid binding protein (I-FABP) was measured by ELISA technique to investigate the integrity of the intestinal barrier. Serum concentrations of I-FABP were significantly higher in psoriasis patients compared with the healthy group and were positively correlated with the presence of bacDNA in patients' plasma. Conclusion: These findings suggest that psoriasis flare up is associated with translocation of bacteria and its metabolites into the blood stream, causing an immune system response. The specific mechanism of bacDNA in the pathogenesis of psoriasis needs to be comprehensively studied. Keywords: psoriasis, gut barrier, I-FABP, skin-gut axes

Interrelationships between Habitual Beverage Consumption, Plasma Biomarkers and Risk of Type 2 Diabetes: Results From a Prospective Case-Control Study

ObjectivesTo elucidate potential pathways of dietary risk of type 2 diabetes (T2D) by defining a model that describes how identified patterns of habitual beverage consumption associate with identified networks of circulating cardiometabolic plasma biomarkers. MethodsThis study included 1,231 cases and 1,560 controls from a nested case-control study of T2D within the Nurses’ Health Study I. Participants completed validated food frequency questionnaires assessing their habitual beverage intake and provided plasma samples to assess 27 different plasma biomarkers of cardiometabolic risk. Common exploratory factor analysis (EFA) was used to identify common factors that separately described beverage consumption patterns and biomarker networks. False discovery rate corrected multivariable-adjusted regression elucidated the relationships between beverage and biomarker factors, and their associations with T2D risk. ResultsEFA revealed five factors describing beverage consumption patterns and seven factors explaining underlying biomarker networks. Preferential consumption of alcoholic beverages was associated with reduced risk of T2D (P < 0.0001) and lower concentrations of detrimental biomarkers of endothelial dysfunction (P = 0.014). Also, a preferential consumption of low-calorie sweetened beverages (LCSBs) was associated with increased risk of T2D (P < 0.0001), and lower concentrations of insulin-like growth factor binding protein 1 and 2, and soluble leptin receptor (P = 0.010). ConclusionsThese analyses provide new mechanistic insight into how different patterns of beverage consumption may relate to T2D risk, and indicate that preferential consumption of LCSB may exert diabetogenic effects through mechanisms involving the disruption of insulin-like growth factor and leptin signalling. Funding SourcesNational Institutes of Health.

MO1007: Intestinal Permeability in Patients After Kidney Transplantation Treated with Two Different Formulations of Extended-Release Tacrolimus

Abstract BACKGROUND AND AIMS A prompt achieving of adequate tacrolimus blood exposure is crucial in early period after kidney transplantation. A gut epithelial barrier integrity may play a role in tacrolimus absorption. The aim of this study was to investigate the intestinal fatty acid binding protein (I-FABP) concentrations as a marker of intestinal permeability in recent kidney transplant recipients (KTRs) treated with two different tacrolimus extended-release formulations. METHOD 48 pairs of consecutive KTRs were analyzed. Within each pair, an early conversion was performed from twice-daily (Prograf) to a once-daily tacrolimus formulation: Advagraf or Envarsus. On the day of discharge from the hospital, I-FABP plasma levels were measured and the tacrolimus concentration-to-dose (C/D) ratio was calculated. Tacrolimus exposure was assessed with an area under the curve (AUC) based on blood trough level and 3-h post-dose measurement. RESULTS There were no differences in recipient age, dialysis vintage, BMI and residual diuresis between the Advagraf and Envarsus groups. The median I-FABP plasma levels [Advagraf: 1450 (750–2100) versus Envarsus 1200 (600–1790) pg/mL; P = .18] and mean tacrolimus AUC [154 (143–164) versus 145 (132–158) ng.h/mL, respectively; P = .30] were similar. As expected, C/D ratio was significantly greater in the Envarsus group [2.06 (1.32–3.09) versus 1.16 (0.72–1.52) in the Advagraf group; P &amp;lt; .001]. Interestingly, log I-FABP correlated inversely with tacrolimus AUC only in the Envarsus group (r = –0.431; P &amp;lt; .01) but not in the Advagraf group (r = 0.05; P = .72). CONCLUSION Our findings suggest that destabilization of the enterocyte membrane integrity may influence the intestinal tacrolimus absorption, which may cause lower tacrolimus exposure in KTRs treated with Envarsus. However, explanation of this phenomenon needs further investigation.

Analysis by transcriptomics and metabolomics for the proliferation inhibition and dysfunction through redox imbalance-mediated DNA damage response and ferroptosis in male reproduction of mice and TM4 Sertoli cells exposed to PM2.5

Sertoli cells play a pivotal role in the complex spermatogenesis process. This study aimed to investigate the effects of PM2.5 on Sertoli cells using the TM4 cell line and a real time whole-body PM2.5 exposure mouse model, and further explore the underlying mechanisms through the application of metabolomics and transcriptomics. The results in vivo and in vitro showed that PM2.5 reduced Sertoli cells number in seminiferous tubules and inhibited cell proliferation. PM2.5 exposure also induced Sertoli cell dysfunction by increasing androgen binding protein (ABP) concentration, reducing the blood-testis barrier (BTB)-related protein expression, and decreasing glycolysis capacity and lactate production. The results of transcriptomics, metabolomics, and integrative analysis of multi-omics in the TM4 Sertoli cells revealed the activation of xenobiotic metabolism, and the disturbance of glutathione and purine metabolism after PM2.5 exposure. Further tests verified the reduced GSH/GSSG ratio and the elevation of xanthine oxidase (XO) activity in the PM2.5-exposed TM4 cells, indicating that excessive reactive oxygen species (ROS) was generated via metabolic disorder caused by PM2.5. Moreover, the redox imbalance was proved by the increase in the mitochondrial ROS level, superoxide dismutase (SOD) and catalase (CAT) activity, as well as the activation of the Nrf2 antioxidative pathway. Further study found that the redox imbalance caused by PM2.5 induced DNA damage response and cell cycle arrest. Additionally, PM2.5 induced ferroptosis through iron overload and lipid peroxidation. Taken all together, our study provided new insights for understanding proliferation inhibition and dysfunction of TM4 Sertoli cells exposed to PM2.5 via metabolic disorder and redox imbalance-mediated DNA damage response and ferroptosis.

Repeated-Sprint Exercise in the Heat Increases Indirect Markers of Gastrointestinal Damage in Well-Trained Team-Sport Athletes.

Athletes engaged in repeated-sprint training in the heat can be at an increased risk of gastrointestinal ischemia and damage in response to a redistribution of blood to working skeletal muscles and the skin. This study investigated the effects of repeated sprinting in hot and cool conditions on markers of gastrointestinal damage. Twenty-five, well-trained, nonheat acclimated male team-sport athletes completed a five-session, repeated-sprint training regimen over 7 days in either HOT (40 °C and 40% relative humidity [RH]) or COOL (20 °C and 40% RH) conditions. Participants underwent a 20-min warm-up and four sets of 5 × 6-s maximal cycling sprints, with 24-s rest and 5-min recovery between sets. Venous blood was collected pre-, post-, and 1hr postexercise and analyzed for intestinal fatty acid binding protein, lipopolysaccharide binding protein, soluble CD14, and heat-shock protein. Intestinal fatty acid binding protein concentrations were significantly increased (p < .004) postexercise (593 and 454pg/ml) and 1hr postexercise (466 and 410pg/ml) on both Days 1 and 5 in HOT. Soluble CD14 increased by 398 and 308ng/ml postexercise (p = .041), and lipopolysaccharide binding protein increased by 1,694ng/ml postexercise on Day 1 in HOT (p < .05) and by 1,520ng/ml on Day 5 in COOL (p = .026). Core and skin temperature, rating of perceived exertion, and thermal sensation were higher (p < .05) in HOT on Days 1 and 5 during sprinting. Repeated sprinting in the heat induced greater thermal strain and mild changes in gastrointestinal damage, likely attributable to the combination of environmental conditions and maximal-intensity exercise.